Monocarboxylate Transporters 1 and 4 and Prognosis in Small Bowel Neuroendocrine Tumors

Simple Summary Monocarboxylate transporters (MCT) are cell membrane proteins transporting lactate, pyruvate, and ketone bodies. For most non-neoplastic cells, the aforementioned energy metabolites are waste products and must be exported via MCTs. However, it seems tumor cells have developed means to shuttle these metabolites through MCTs and utilize them for energy production. MCTs contribute to the acidification of the tumor microenvironment and are associated with drug resistance making them potential therapeutic targets. MCT expression has been shown to correlate with prognosis in a range of human solid tumors. In small bowel neuroendocrine tumors, the expression of MCTs 1 and 4 is reported for the first time. High MCT4 expression is associated with improved prognosis. Abstract Monocarboxylate transporters (MCTs) are cell membrane proteins transporting lactate, pyruvate, and ketone bodies across the plasma membrane. The prognostic role of MCTs in neuroendocrine tumors is unknown. We aimed to analyze MCT1 and MCT4 expression in small bowel neuroendocrine tumors (SB-NETs). The cohort included 109 SB-NETs and 61 SB-NET lymph node metastases from two Finnish hospitals. Tumor samples were immunohistochemically stained with MCT1 and MCT4 monoclonal antibodies. The staining intensity, percentage of positive cells, and stromal staining were assessed. MCT1 and MCT4 scores (0, 1 or 2) were composed based on the staining intensity and the percentage of positive cells. Survival analyses were performed with the Kaplan–Meier method and Cox regression, adjusted for confounders. The primary outcome was disease-specific survival (DSS). A high MCT4 intensity in SB-NETs was associated with better DSS when compared to low intensity (85.7 vs. 56.6%, p = 0.020). A high MCT4 percentage of positive cells resulted in better DSS when compared to a low percentage (77.4 vs. 49.1%, p = 0.059). MCT4 scores 0, 1, and 2 showed DSS of 52.8 vs. 58.8 vs. 100% (p = 0.025), respectively. After adjusting for confounders, the mortality hazard was lowest in the patients with a high MCT4 score. MCT1 showed no association with survival. According to our study, a high MCT4 expression is associated with an improved prognosis in SB-NETs.

Introduction
Neuroendocrine tumors (NET) are neoplasms that can develop basically in any organ system where endocrine cells are present [1]. Prognosis varies in relation to anatomic site and tumor grade [2]. Gastroenteropancreatic NETs (GEP-NET) are among the most common ones, developing from the enteroendocrine cells of the gastrointestinal tract and pancreas [3,4]. The incidence rate of all GEP-NETs is 3.56 per 100,000 persons per year as reported by the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) Program. The incidence of small bowel NETs (SB-NET) is approximately 1.05 per 100,000 person-years [3]. The Surveillance of Rare Cancers in Europe (RARECARE) project estimated the European incidence of well-differentiated non-functioning GEP-NETs to be 11 per 1,000,000 person-years and 0.23 per 1,000,000 person-years for well differentiated functioning GEP-NETs [4]. NET incidence has increased 6.4-fold over the past decades, although this increase is at least partly due to increased number of upper gastrointestinal tract endoscopies and radiological imaging studies [3]. SB-NETs are among the ones with worst survival, but still due to the natural history of this neoplasia, deaths during Grade I differentiation occur during a very long follow-up with a median survival around 14 years [3]. The requirement of a long follow-up causes difficulties in the clinicopathological research of SB-NETs.
In most non-neoplastic cells, energy is mainly produced through aerobic mitochondrial metabolism, whereas in rapidly growing cells, such as cancer cells, energy is often produced by anaerobic glycolysis [5]. This results in high lactate and carboxylic acid levels [5]. pH regulation is therefore needed, and this is controlled in part by monocarboxylate transporters (MCTs) [6]. MCTs are a cell membrane transporter family encoded by SLC16 genes. The family comprises 14 different proteins, MCTs 1-4 being the most investigated ones [7]. MCT1 (SLC16A1) transports lactate into the cell but in some physiologic conditions it can contribute to lactate efflux. MCT4 (SLC16A3) is a key mediator in lactate efflux [8]. These MCT receptors have been associated with drug resistance, and are among potential therapeutic targets for treating solid tumors [9,10]. MCT1 inhibitors are in the advanced development phase and MCT4 inhibitors still in the discovery phase [11]. The expression patterns of MCT1 and MCT4 have been previously studied in many cancers, including gastric, lung, colorectal, and esophageal cancers [6,[11][12][13][14]. However, according to our knowledge, no studies assessing the role of MCTs in neuroendocrine tumors or SB-NETs exists in clinical cohorts. A single study of mouse pancreatic NETs exist, which suggests that MCTs are upregulated in hypoxic conditions and may have a role in NETs as well [15].
In this study, MCT1 and MCT4 were studied for the first time in relation to SB-NET prognosis. MCTs have a significant role in the adaptation to acidosis. Acidosis, in turn, is suggested to contribute to cancer progression and aggressiveness. This suggests that MCTs could potentially affect prognosis, making MCTs 1 and 4 interesting research targets. The aim was to test the prognostic significance of MCT1 and MCT4 in SB-NETs in a large retrospective consecutive series of SB-NETs from two institutions in Northern and Central Finland with very long follow-up and complete disease-specific survival data.

Patients and Data Collection
The initial cohort and data collection has been previously described [16]. This study included 125 patients treated for ileal and jejunal SB-NETs in Oulu University Hospital and Central Finland Central Hospital. The patients in Oulu were treated between 9 February 2000 and 7 February 2018 and in Jyväskylä between 24 February 2000 and 31 December 2017. SB-NET lymph node metastases were detected in 95 of these patients and lymph node samples were also included in these cases. The collection of clinical patient data was possible through electronically stored archives. The Cause of Death Registry maintained by Statistics Finland provided the survival data. The end of follow-up was 31 December 2019. The Oulu University Ethics Committee approved this study (EETTMK 81/2008). The National Authority for Medicolegal Affairs (VALVIRA) waived the need for informed consent and approved for the use of data and samples.
Experienced pathologists determined the histological diagnoses at the time of treatment. The 8th edition of the AJCC/UICC TNM categories was used to determine tumor stage and the WHO 2019 classification of tumors of the digestive system was used to determine the tumor grade [17,18].

Immunohistochemical Staining
Tissue samples from primary tumors and lymph node metastases were fixed in formalin and embedded in paraffin at the time of diagnosis. Representative samples including those of the deepest tumor invasion were identified based on diagnostic hematoxylin-eosin slides. For immunohistochemistry, the sample blocks were retrieved from archives and cut into tissue sections of 3.5 µm in thickness. The sections were deparaffinized in xylene and rehydrated through graded alcohols. Antigen retrieval was performed with citrate buffer at pH 6 in a microwave oven, first at 800 W for 2 min and then at 150 W for 10 min. Tissue sections were cooled at room temperature for 20 min and rinsed both in distilled water and in phosphate-buffered saline containing Tween (PBS-Tween). Endogenous peroxidase activity was neutralized in peroxidase-blocking solution (Dako, Glostrup, Denmark, S2023) for 5 min, followed by two 5-min wash cycles in PBS-Tween. After this, sections were incubated with mouse monoclonal antibodies in dilute solution (Dako S2022); MCT1 for 60 min (diluted 1:100, Santa Cruz Biotechnology (H-1): sc-365501), MCT4 for 60 min (diluted 1:500, Santa Cruz Biotechnology (H-90): sc-50329). After another two 5-min wash cycles in PBS-Tween, samples were incubated with En-Vision polymer (Dako K5007) for 30 min and again washed in PBS-Tween for two cycles of 5 min. After the final wash, diaminobenzidine working solution (Dako K5007) was used as a chromogen. Lastly, the samples were rinsed in distilled water and counterstained in hematoxylin for 1 min. All staining was done with the Dako Autostainer (Dako, Copenhagen, Denmark). Cancer tissues with a high expression of MCT1 and MCT4 were used as external positive controls. To confirm the antigen preservation in the old paraffin blocks, we compared the MCT1 and MCT4 staining intensities in SB-NETs between old and new blocks divided by the median age of the blocks. No significant differences were found.

Immunostaining Assessment
Microscopic slides were scanned at 20× magnification using an Aperio AT2 digital slide scanner.
The immunoreactivity of MCT1 and MCT4 was analyzed by two independent researchers (JR and HH) who were completely blinded to the clinical data and did not participate in the statistical analyses. We assessed the intensity of staining (0-3), the percentage of positive cells (0-100), and the percentage of membrane-positive cells (0-100). The tumor stromal staining pattern was assessed as 0, no detectable staining; 1, focal staining; 2, areas of diffuse staining present in less than half of stromal area; 3, expression of moderate density distributed in more than half but not in all parts of the tumor stroma; 4, dense expression extending throughout the stroma as previously described [19]. Mean values of two independent estimates were used if there was no difference over 1 in the intensity or over 30% in the percentage. If the difference was more extensive, consensus was reached after re-evaluation. Re-evaluation was needed only in 3 cases. To avoid results found by chance or multitesting, predefined cut-offs for intensity (0-1 defined as low, and 2-3 as high), percentage (0-99 as low, and 100 as high), and stromal staining (0 as negative and 1-3 as positive) were used. Furthermore, based on both the intensity and percentage of positive cells, MCT1 and MCT4 scores were calculated as follows. A low intensity (0-1) was awarded zero points, and a high intensity (1-2) one point. Similarly, low percentages (0-99) were awarded zero points and high percentage (100) for one point. The intensity and percentage points were summed up, resulting in an MCT score of 0 (both low), 1 (low and high), or 2 (both high).

Statistical Analysis
For comparison of baseline values, The Chi-squared test for categorized variables and the Mann-Whitney U-test for continuous variables was used. Survival times were calculated from the date of operation until the time of death or the end of follow up. Disease-specific survival rates (DSS) were calculated using the Kaplan-Meier method with a log-rank test stratified by MCT intensity (low/high), percentage (low/high), stromal staining (low/high), and score (0, 1 and 2). Crude and adjusted hazard ratios (HR) for mortality were calculated using Cox regression models. Cox regression was adjusted for age, sex, stage (I-II, III, IV), the grade of differentiation (G1 or G2), adjuvant somatostatin therapy (no/yes) and sample type (surgical resection specimen or biopsy). A p-value of less than 0.05 was considered significant. The statistical analyses were performed with IBM SPSS statistics 27 for Windows (IBM Corporation, Armonk, NY, USA).

Patients
Of the whole cohort, including 125 SB-NETs and 95 SB-NET lymph node metastases, adequate samples and representative immunostainings were available for 109 patients (MCT1) and 104 patients (MCT4). The final cohort included 61 lymph node metastases for both MCT1 and MCT4 evaluation.
Of the 109 SB-NET patients, total of 60 (55.0%) were men and the median age was 66 (IQR 56-72) years. Of tumors, 12 (11.0%) were stage I-II, 59 (54.1%) were stage III, and 38 (34.9%) were stage IV. Additionally, 85 (78.0%) were Grade I and 24 (22%) were Grade II whereas no Grade III tumors or neuroendocrine carcinomas (NEC) were operated for in either of the hospitals involved during the study period. Median follow-up time was 66 (IQR 40-119) months. Thirty-five patients died during follow-up, of which 20 deaths were considered to be disease-specific. Baseline characteristics are presented separately for patients with available MCT1 (Table 1) and MCT4 (Table 2).

MCT1 and MCT4 Staining
The MCT1 intensity median was 2.0 (IQR 1-2.75) and a total of 15 (13.8%) cases showed a staining intensity of 0. The MCT1 percentage median was 100 (IQR 100-100) with as much as 84 (77.1%) cases showing 100% staining. Examples of immunohistochemical staining are presented in Figure 1. MCT1 in tumor stroma was seen only in three cases and further analyses were not performed. All lymph node metastases showed negative MCT1 staining (even though external control was clearly positive).

MCT1 and MCT4 Association with Clinicopathological Variables
Associations of the MCT1 score with clinicopathological variables are presented in Table 1. Only the tumor location in the jejunum showed a statistically significant

MCT1 and MCT4 Association with Clinicopathological Variables
Associations of the MCT1 score with clinicopathological variables are presented in Table 1. Only the tumor location in the jejunum showed a statistically significant association with a lower MCT1 score (p = 0.033). The MCT4 score showed no statistically significant associations with the studied variables ( Table 2).

MCT1, MCT4, and Survival
Disease-specific survival in the whole cohort at 5 years was 91.2%, at 10 years it was 74.3%, and during the whole follow-up it was 64.8%. Overall survival at 5 years was 84.4%, at 10 years it was 61.3%, and during the whole follow-up it was 41.0%.
Neither the MCT1 intensity, percentage of positive cells, or the MCT1 score were associated with DSS (Table 3, Figure 2A). Table 3. Disease-specific survival rates based on MCT1 and MCT4 stainings (intensity, percentage, MCT score, and stromal staining) in both primary tumors and lymph node metastases. Lymph node metastases showed negative MCT1 staining and therefore these are not presented.

Disease-Specific Survival
No. A high MCT4 intensity was associated with better DSS when compared to low intensity (85.7 vs. 56.6%, p = 0.020) ( Figure 2B). A high MCT4 percentage of positive cells resulted in a higher DSS when compared to a low percentage (77.4 vs. 49.1%, p = 0.059), although this was without statistical significance ( Figure 2C). The MCT4 score of zero showed worse survival when compared with the MCT4 score of one and the MCT4 score of two (52.8 vs. 58.8 vs. 100%, p = 0.025) ( Figure 2D). Survival percentages are presented in Table 3.
For future studies we aim to find the optimal cut-off values for MCT1 and MCT4 expression; we provided ROC curves of the MCT1 and MCT4 variables, as presented in Table 3. Optimal cut-offs based on the ROC curves showed only minor differences when compared to the currently used cut-off values (Supplementary data, Supplementary Figures S1-S3).
Crude and adjusted HRs for disease-specific mortality are shown in Table 4. Adjusted HR in the high-MCT4-intensity group was 0.19 (95%CI 0.03-1.48) and in the high-MCT4percentage group it was 0.23 (95%CI 0.09-0.61) when compared to low expression (Reference). MCT4 score showed a linearly lower adjusted mortality hazards with an increasing score (Score 1 HR 0.41; 95%CI 0.16-1.10 and Score 2 HR 0.00; 95%CI 0.00-0.00) when compared to Score 0 (Reference), although since no events occurred in the Score 2 group, HR could not be estimated.

Discussion
In this study, high MCT4 expression in SB-NETs was associated with an improved prognosis both in a univariate model and after adjusting for confounding factors. MCT1 expression was not associated with prognosis. In SB-NET lymph node metastases, MCT1 or MCT4 expression showed no prognostic value.
Our present study has several strengths. This study involved 109 patients, representing one of the largest published cohorts of SB-NETs with representative paraffin-embedded samples and complete clinical data. For the first time, MCT1 and MCT4 expression patterns were evaluated in SB-NETs, including 61 SB-NET lymph node metastases, and were examined in relation to clinicopathological variables and prognoses. We used whole section slides, including a selection of samples with the deepest invasion area. Our study included a long follow-up time, which is particularly important in relatively slowly progressing diseases like SB-NETs. Nearly 100% complete registry data with information on causes of death from Statistics Finland was used. This allowed for DSS analyses, which are of upmost importance in cancers with a good prognosis. Still, our study has some limitations. SB-NETs are rare neoplasms and despite including patients from a period of almost two decades, the sample size was relatively small, especially regarding the number of cancer deaths. Strong conclusions are possible only after replication studies. However, the association of high MCT4 expression and a good prognosis seems to be a real finding, with staining intensity, percentage, and score point estimates showing similar results both in uni-and multivariate models. According to the current analysis, mortality risk decreases with increasing MCT4 staining intensity and the percentage of positive cells, and future studies will need to set an optimal cut-off. Because of the long time period in the current study, the treatment and diagnostics of these tumors have possibly been improved, which can be a confounding factor. Although being non-selected for, the study population only consisted of G1 and G2 NETs, making data more homogenous and reliable. It should be noted that these results are not applicable for G3 NETs or NECs. A conservative approach has been favored in both study centers concerning G3 NETs and NECs as suggested in the ESMO guidelines [20].
SB-NET prognosis has mainly been determined by traditional factors such as Ki-67 proliferation index (Grades) and TNM stage. A nomogram including several different variables has also been developed by Modlin et al. in order to better predict the prognosis of individual patients [21]. At least three other studies have later tried to validate the nomogram for clinical use [22][23][24]. In addition to tumor-derived variables, continuous variables such as age, plasma CgA, and urinary 5-HIAA are used in the nomograms. Other clinical variables such as WHO performance status (PS) have been proved to be important in cancer surgery and PS probably affects SB-NET surgery outcomes similarly [25]. The validation study by Levy et al. found WHO performance status two to be an independent prognostic factor for a worse DSS in SB-NETs [24]. MCT4 has potential to improve the accuracy of survival prediction as it adds to the list of new emerging prognostic factors and biomarkers to predict disease progression and survival of SB-NET patients. MCT status could be included in these nomograms if the results of our current study are later replicated.
Acidosis, hypoxia, and glycolytic metabolism are associated with cancer cell viability and their ability to progress [26]. An intense proliferation of cancer cells results in hypoxia related to the growing distance to the microvasculature [27]. The resulting pH arrangement is characteristic for malignant tumors, resulting in extracellular acidification. The Warburg effect is a theory suggesting that cancer cells tend to switch to glycolytic metabolism even in normoxic conditions (aerobic glycolysis), producing large amounts of lactate [28]. To avoid intracellular acidification, lactate must be expelled to the extracellular space where lactate is not only a waste for tumors. It has been proposed that cancer cells become more aggressive and resistant to therapy in an acidifying microenvironment [29]. MCTs play a significant role in glycolysis and the adaptation to acidosis [5] and theoretically, according to the Warburg hypothesis, a high expression could be related to a poor prognosis, also making MCTs potential therapeutic targets [5].
MCT1 expression has been evaluated in several previous studies of certain human solid tumors. In a large cohort study of operated gastric cancer patients, MCT1 expression was associated with a higher T-class, but not with prognosis [12], and in esophageal adenocarcinoma, a high MCT1 expression was associated with an improved prognosis [6]. High MCT1 expression levels have been reported in, for example, breast and lung cancer [30,31]. In one gastric cancer study, an MCT1 inhibitor increased chemotherapy sensitivity, implying that targeting MCT1 may have therapeutic potential [9]. Still, no clear consensus in any solid tumor exists regarding MCT1 expression and prognosis. In SB-NETs, no previous studies exist. In the current study, the result seems to follow previous studies on several other solid tumors with no prognostic significance. It is noteworthy that despite adequate positive controls, lymph node metastases showed no MCT1 positivity, suggesting that only MCT1-negative clones were prone to metastatic behavior. Still, no survival difference in primary tumors was seen to be related to MCT1 expression.
In the aforementioned large gastric cancer cohort study, MCT4 was associated with positive lymph node status but again, not with prognosis [12]. In esophageal cancer, high MCT4 expression was associated with a poor prognosis [6], and similar results were found in previous studies on lung, colon, and prostate cancers [31][32][33]. Overall, in previous studies on solid tumors, MCT4 was commonly associated with a poor prognosis. Conversely to these findings, in the current study, which reports results for the first time in SB-NET, high MCT4 expression associated with improved survival.
The most studied function of MCT1 is in mediating lactate influx, however, in altered physiological conditions, MCT1 can also mediate lactate efflux [8]. MCT4 in turn is mostly expressed in highly glycolysis-dependent tissues where it mediates lactate efflux [8]. Lactate shuttling is an interesting phenomenon in cancers and various theories have been proposed to describe it [34][35][36][37]. This bi-directional shuttling of lactate seems to have an intricate role in the development, growth, and metastasis of cancer [38]. One of these theories, the reverse Warburg effect, theorizes that cancer cells can create a pseudo-hypoxic microenvironment in the stroma, inducing MCT4 upregulation and increased glycolysis in stromal fibroblasts through HIF-1α activation. The fibroblasts then efflux lactate through MCT4, after which lactate is imported by tumor cells through MCT1 and used as an oxidative metabolite [34]. Through MCT1 or MCT4 inhibition, this pathway could potentially be interrupted, leading to cancer cell starvation.
NETs are among the most extensively vascularized cancers, with an intratumoral vessel density approximately 10-fold higher when compared with carcinomas. Intratumoral microvascular density is the highest in low-grade tumors and it is associated with an improved prognosis and prolonged survival [39]. GEP-NETs express several proangiogenic factors including vascular endothelial growth factor (VEGF), which is the main driver of angiogenesis [40]. VEGF expression is strictly modulated by oxygen levels through HIF-1α [41]. Considering these GEP-NET-specific features, it seems plausible that increased hypoxia and acidotic tumor microenvironments could cause an increased activation of proangiogenic factors further increasing intratumoral microvascular density. Since the majority of our cases were G1, with some G2 NETs, this could cause favorable prognosis effects and could be one of the mediating factors explaining the high MCT4 activity and good prognosis.
The current study has some potential clinical implications. MCT proteins are potential targets for therapeutic purposes. Cancer cells are known to become more aggressive and treatment-resistant in an acidifying tumor microenvironment. Because of their role in controlling the acidity of the tumor microenvironment, MCTs could be important factors in regulating cancer progression. Understanding how MCT expression is tied to patient prognosis can help develop targeted therapies to slow down cancer progression or even therapies that lead directly to cancer cell starvation and cell death. Our study provides important information on the prognostic value of MCT1 and MCT4 in SB-NETs, showing the possibility of contrary results (regarding MCT4 expression) and heterogeneity between different cancers. Replication studies are, however, needed.

Conclusions
In our study, we reported the presence of MCT1 and MCT4 in SB-NETs. High MCT4 expression was associated with improved survival also after adjusting for confounders.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/cancers14102552/s1, Figure S1: ROC curve analysis of disease-specific survival related to MCT1 intensity and MCT1 percentage in primary tumors showing no prognostic significance with any cut-off value, Figure S2: ROC curve analysis of disease-specific survival related to MCT4 intensity, MCT4 percentage and MCT4 stroma in primary tumors proposing optimal cut-off (furthest point from reference line) values of 2 in staining intensity and >95 in percentage of positive cells. MCT4 stroma showed no prognostic significance with any cut-off value, Figure S3: ROC curve analysis of diseasespecific survival related to MCT4 intensity, MCT4 percentage and MCT4 stroma in lymph node metastases proposing optimal cut-off (furthest point from reference line) values of 2.5 in staining intensity and >85 in percentage of positive cells. MCT4 stroma crossed the reference line and showed no prognostic significance.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the hospital districts. Use of the samples and patient data were approved by the Ethics Committee (EETTMK: 81/2008) and by the National Authority for Medicolegal Affairs (VALVIRA).

Informed Consent Statement:
The need for written informed consents was waived by National Authority for Medicolegal Affairs (VALVIRA) due to its retrospective nature.

Data Availability Statement:
Anonymized data is available upon request from the corresponding author.


Physiological role of isocitrate lyase in dibenzo-p-dioxin and dibenzofuran metabolism by Sphingomonas wittichii RW1

Background Sphingomonas wittichii RW1 is one out of three strains capable of metabolizing dioxin as a sole source for carbon and energy. Under laboratory conditions the degradation rates for these aromatics are relatively high (5 and 8 h for dibenzofuran (DBF) and dibenzo-p-dioxin (DD), respectively). However, their degradation rates are much lower in the environment due to several factors. One of these factors is the availability of other carbon sources. Acetate is a metabolized carbon source by S. wittichii RW1 and its presence in the environment would have a negative impact on DBF and DD degradation. In addition, expression of most of the genes for DBF and DD degradation were downregulated when grown on acetate compared to their growth on DBF and DD. We hypothesized that blocking the acetate utilization pathway in S. wittichii RW1 would prevent it from using acetate when present along with DD and DBF in contaminated sites. Results Blocking the glyoxylate shunt by deleting isocitrate lyase gene (icl) prevented the mutant strain (RW1Δicl) from using acetate as a sole carbon source thus depending on available DBF and DD in polluted sites. Our results showed that deletion of icl did not affect growth of S. wittichii RW1 on DBF and DD but blocked it from growing on acetate. Conclusion Our results introduces an engineered strain that can be used as a new candidate to clean dioxin-contaminated sites which are rich with acetate.

Background
Sphingomonas wittichii RW1 is one out of three strains capable of metabolizing dibenzo-p-dioxin as a sole source for carbon and energy, accordingly, has received extensive research from many scientists worldwide [3,16]. Dioxins are a group of chemically related aromatics that include polychlorinated dibenzo-p-dioxins and dibenzofurans. These compounds are chemically inert, recalcitrant, lipid soluble, and toxic to humans and animals [17]. Dioxins are released to the environment as by products from the manufactory of pesticides and herbicides, incineration of wastes, or can be produced naturally from forest fires and volcanoes eruption; therefore, dioxins have become ubiquitous pollutants in the environment [21]. Reports have stated that more than 50% of the dioxins excluded to the environment is from incineration of municipal solid waste. These persistent compounds remain in the sediments for decades which is why it is necessary measure dioxin concentration if waste water effluent [19].
Degradation rates of S. wittichii RW1 for dibenzop-dioxin (DD) and dibenzofuran (DBF) under laboratory condition are relatively high, 5 and 8 h for DD and DBF, respectively, when grown on 5 mM of each substrate [24]. However, their degradation capabilities in the environment are restricted to nutrient availability, generation of Faisal and Rasol Journal of Genetic Engineering and Biotechnology (2022) 20:52 dead end products, trace concentrations of dioxins, water activity, and low adaptation to soil environment [4,5].
Attempts to engineer S. wittichii RW1 to improve its degradation capabilities have succeeded by introducing membrane superchannels from Sphingomonas sp. A1. These superchannels facilitated the incorporation of polyaromatic hydrocarbons into S. wittichii RW1 cells thus enhanced the degradation rate of hazardous compounds in contaminated sites [1]. Interestingly, genome analysis of S. wittichii RW1 revealed that this strain contained genes that carried the ability to perform all the steps included in the biphenyl degradation pathway except the dehydrogenation of cis 2,3-dihydro-2,3-dihydroxybiphenyl and this was the only missing gene that prevented it from growing on biphenyl. Engineering this strain by introducing an appropriate dehydrogenase from a biphenyl degrader enabled S. wittichii RW1 to grow on biphenyl at relatively high rates [8].
Acetate is an important carbon source and easy to use compound. It is both aerobically and anaerobically metabolized by bacteria. As a result, inhibiting such microbial activities leads to acetate accumulation in soil. Acetate concentrations varies according to type of soil being the highest in salt-marsh sediments that ranges between 0.1 and 1.0 mM [10]. Acetate is produced in soils by several pathways, it can be produced anaerobically by bacteria via acetogenesis or through anaerobic degradation of cellobiose [12]. Evidence for release of acetate from fine roots and associated mycorrhiza have also been detected [11]. Acetate is a preferable carbon source by S. wittichii RW1 and its presence in the environment would have a negative impact on DD and DBF degradation. In addition, expression of most of the genes for DD and DBF degradation is down regulated when grown on acetate compared to their growth on DD and DBF [9]. Oxygen uptake by S. wittichii RW1 resting cells grown on acetate and subjected to DD and DBF was relatively less compared to resting cells on DD and DBF [24].In addition, acetate has been reported to inhibit the degradation rate of some polyaromatic hydrocarbons (PAH) in the environment probably due to its preference by PAH degrading microorganisms [20]. The presence of more than one carbon source in the environment where one is more favorable than the other leads to diauxic growth where the organism cannot metabolize the unfavorable compound until the favorable one is totally consumed [13]. Therefore, we hypothesized that blocking the acetate utilization pathway in S. wittichii RW1 would prevent it from using acetate when present along with DD and DBF in contaminated sites. Engineered strains of S. wittichii RW1 will obtain all their carbon source from contaminated aromatics instead of utilizing acetate. Few studies have been conducted on improving biodegradation capabilities in S. wittichii RW1, however, improving biodegradation capabilities by engineering metabolic pathways has not been reported elsewhere.

Bacterial strains and plasmids
S. wittichii RW1 was kindly provided by Dr. Gerben Zylstra at Rutgers University (New Jersey, USA). Escherichia coli DH5α was used as the recipient in transformation experiments (Invitrogen, Waltham, MA). The cloning vector pGEM-7Z(+) (Promega, Madison, Wisconsin) was used to clone icl gene containing fragment. The low copy number plasmid pRK415 was kindly provided by Dr. Donald Kobayashi at Rutgers University (New Jersey, USA) and used to construct the final knockout clone and deliver it to strain RW1 to promote gene disruption.

Molecular techniques and DNA manipulations
Ultraclean microbial DNA isolation kit (MoBio, Carlsbad, CA) was used to isolate genomic DNA from S. wittichii RW1 by following the protocol supplied. Plasmid DNA was extracted from E. coli DH5α using Nucleospin plasmid (NoLid) purification kit (Macherey-Nagel, Germany) and following the recommendations provided. General molecular techniques including restriction enzyme digestion, gel electrophoresis, ligation, and transformation were performed according to Sambrook et al. [22]. DNA fragments were amplified using the Phusion high fidelity PCR (polymerase chain reaction) kit (New England Biolabs) and purified from PCR solution or 1% agarose gel when necessary by QiaexII extraction kit (Qiagen). DNA sequence of the final clone introduced in strain RW1 was sequenced to verify that it lacked undesired mutations. Sequencing was done through GENEWIZ sequencing company (New Jersey, USA).

Culturing of S. wittichii RW1
In growth curve experiments, S. wittichii RW1 was grown in 250 ml flasks containing 50 ml MSB medium (Mineral Salts Basal medium) and incubated at 28 o C with shaking (180 rpm). MSB was supplemented with acetate (10 mM), DBF (3 mM), and DD (3 mM) as sole carbon sources individually. Acetate was added as sodium acetate salt while DBF and DD were added as crystals. Growth curve experiments were conducted in triplicates and optical density readings were recorded at 2 h intervals. Growth curves were drawn using GraphPad Prism (v. 8.0.2), and all statistics including calculation of average and standard deviation were automatically calculated by Prism and shown on the growth curves.
For the purpose of searching for gene knockout strains, S. wittichii RW1 containing the knockout vector (shown below) was grown in 5 ml Luria-Bertani broth (LB) and and was then sequenced to confirm the presence of the desired gene and check for the integrity of the sequence. The knockout vector was delivered to S. wittichii RW1 (wild type) and transferred for several times on 5 ml LB broth as mentioned above. Colonies that were kanamycin resistance and tetracycline sensitive (conducted double homologous recombination which replaced the wild type copy of icl with the disrupted copy of icl constructed on pRK415) were isolated and confirmed by PCR of the fragment containing icl and compared to the wild type.

Effect of isocitrate lyase deletion on acetate utilization
After several transfers on LB media, several transconjugants were isolated that had experienced double homologous recombination between the wild type copy of icl gene and its disrupted copy on the knockout vector. Results were confirmed by PCR using the primers icl-F and icl-R as shown in Fig. 1. As expected, deletion of icl from S. wittichi RW1 abolished this bacterium from growing on acetate as a sole carbon source (Fig. 2). Complementation experiments conducted by transforming RW1Δicl with a pRK415 vector carrying an intact copy of icl reverted the transformed knockout strain to its wild-type state (data not shown).

Effect of isocitrate lyase deletion on DBF and DD degradation
Growth curve experiments on DBF and DD were done for S. wittichii RW1Δicl and compared to the growth of the wild type strain. Fortunately, no differences in growth rates was observed between the knockout strains and the wild type (Fig. 3) indicating that icl deletion has no effect on the capability of S. wittichi RW1 to metabolize DBF and DD and this strain could be used as a suitable candidate to remediate DD and DBF polluted sites that are rich in acetate.

Discussion
Isocitrate lyase along with malate synthase are two key enzymes in bacteria for the glyoxylate shunt pathway. Isocitrate lyase catalyzes the split of isocitrate to glyoxylate and succinate while the former is converted to malate via malate synthase (Fig. 4). The glyoxylate shunt enables bacteria to grow on acetate as it bypasses the loss of the two carbons in acetate as carbon dioxide in the TCA cycle [18].
Deleting icl from RW1 lost the ability of RW1Δicl to utilize acetate as a sole carbon source while the wild type phenotype was reverted when an intact copy of icl was introduced to RW1Δicl. These results all together identify the importance of icl in the metabolism of acetate and possibly all other two carbon containing molecules and provides evidence for the presence of a single copy of icl in this bacterium. In addition, complementation experiments verify that the phenotype detected was due to icl deletion and not to a polar effect of downstream genes. No previous studies have deleted icl from S. wittichii RW1; however, site-directed mutagenesis of this gene in E. coli have showed the importance of four histidine residues. The highest effective mutation was seen with H-184. Substitution of H-184 to lysine, arginine, or leucine lead to total loss of enzyme activity while the mutated protein with H-184 substituted with glycine retained 28% of its activity. Protein gel electrophoresis of these mutated proteins showed weak assembly of the Icl subunits into the tetrameric active protein [6].
Due to the fact that icl deletion had no effect on the degradation of DBF and DD, our results shows that this mutant strain is a suitable candidate for remediating such toxic aromatics from environments that are rich in acetate. Interestingly, acetate is one of the intermediates formed from the downstream degradation pathway of DBF and DD and represents 2 out of 12 carbon atoms of their structure (Fig. 5). However, blocking the mutant from using acetate had no effect on its growth rate on these aromatics the reason is probably due to the use of acetate formed from the degradation pathways in the synthesis of fatty acid and other essential building blocks in the cell. S. wittichii RW1 has shown to manage gene regulation for DD and DBF degradation according to its surroundings. A recent study showed that this bacterium adapted the changes in DD availability when DD was absorbed to smectite clay mineral saponite by upregulating genes that were thought to be involved in the uptake and chemotaxis of DD [2]. In E. coli acetate can be metabolized to acetyl-coA through two major pathways: the reversible Pta-AckA pathway and the irreversible acetyl-coA synthetase pathway. Feeding E. coli growing in log phase with 13 C-acetate resulted in the detection of labeled metabolites not only of TCA intermediates but from all central carbon metabolites of carbohydrates [7].
A different study showed that effects of adding acetate decreased the degradation rate of several pharmaceuticals due to feedback inhibition of acetate concentrations which comes in an agreement with our study [14]. These results support our idea for acetate flow and indicates that acetate can be channeled through different pathways in bacteria as expected. In addition, environmental bacteria including S. wittichii RW1 when present in the environment faces carbonlimited conditions which has been shown to induce the expression of polyaromatic hydrocarbon degrading genes [23]. As a results, our mutant strain that is incapable of using acetate will be continually facing carbonlimited conditions and will have improved DD and DBF degrading capabilities. However, the presence of other acetate metabolizers in the environment might be considered as a limitation of this study. Future studies are needed to confirm our work along with growth curve experiments showing the disappearance of DD and DBF when acetate is present or absent. Recent studies have incorporated the use of omics in biodegradation studies for better understanding of how microbes interact with each other in the environment [15]. Such experiments may be useful to identify how the presence of other organisms in contaminated area may affect DD and DBF degradation.

Conclusion
This research was successfully capable of producing an engineered strain of S. wittichii RW1 that has a metabolic block in the glyoxylate shunt through deletion of isocitrate lyase gene. The mutant RW1Δicl was no longer capable of growing on acetate as sole carbon source compared to the wild type. However, RW1Δicl capability to metabolize DBF and DD was not affected. This strain will be a perfect candidate to metabolize such aromatics in contaminated sites rich with acetate.


s2fieldsofstudy: Biology Chemistry

text:
DOPAnization of tyrosine in α-synuclein by tyrosine hydroxylase leads to the formation of oligomers

Parkinson’s disease is a progressive neurodegenerative disorder characterized by the preferential loss of tyrosine hydroxylase (TH)-expressing dopaminergic neurons in the substantia nigra. Although the abnormal accumulation and aggregation of α-synuclein have been implicated in the pathogenesis of Parkinson’s disease, the underlying mechanisms remain largely elusive. Here, we found that TH converts Tyr136 in α-synuclein into dihydroxyphenylalanine (DOPA; Y136DOPA) through mass spectrometric analysis. Y136DOPA modification was clearly detected by a specific antibody in the dopaminergic neurons of α-synuclein-overexpressing mice as well as human α-synucleinopathies. Furthermore, dopanized α-synuclein tended to form oligomers rather than large fibril aggregates and significantly enhanced neurotoxicity. Our findings suggest that the dopanization of α-synuclein by TH may contribute to oligomer and/or seed formation causing neurodegeneration with the potential to shed light on the pathogenesis of Parkinson’s disease.

Parkinson's disease is a progressive neurodegenerative disorder characterized by the preferential loss of tyrosine hydroxylase (TH)-expressing dopaminergic neurons in the substantia nigra. Although the abnormal accumulation and aggregation of α-synuclein have been implicated in the pathogenesis of Parkinson's disease, the underlying mechanisms remain largely elusive. Here, we found that TH converts Tyr136 in α-synuclein into dihydroxyphenylalanine (DOPA; Y136DOPA) through mass spectrometric analysis. Y136DOPA modification was clearly detected by a specific antibody in the dopaminergic neurons of α-synuclein-overexpressing mice as well as human αsynucleinopathies. Furthermore, dopanized α-synuclein tended to form oligomers rather than large fibril aggregates and significantly enhanced neurotoxicity. Our findings suggest that the dopanization of α-synuclein by TH may contribute to oligomer and/or seed formation causing neurodegeneration with the potential to shed light on the pathogenesis of Parkinson's disease. Altered motor symptoms caused by Parkinson's disease (PD) are mainly attributable to the preferential loss of dopaminergic neurons in the substantia nigra (SN) 1,2 . The α-synuclein (αSyn) gene is considered a major genetic contributor of this disease. Several point mutations, such as E46K or A53T, and genomic multiplications in the αSyn gene have been linked to familial PD [3][4][5][6][7] . αSyn is a natively unfolded protein constituted by 140 amino acids with three distinct regions including an amphipathic N-terminal region (1-60), a hydrophobic nonamyloid-β component region (61-95) and a highly acidic C-terminal region (96-140) 8 . It is physiologically involved in neurotransmitter release at presynaptic terminals and in microtubule regulation as a microtubule-associated protein (MAP) [9][10][11] . In PD brain, αSyn forms an abnormal aggregation that is the major component of Lewy pathology 12 . This aggregation has a conformational polymorphism that varies from a small oligomer to an amyloid fibril, which generates biochemical differences in neurotoxicity 13,14 . In particular, recent studies indicate that the αSyn oligomeric species induce more pathogenic effects and play a key role in PD [15][16][17] . Interestingly, even though αSyn is abundantly expressed in neurons throughout the entire brain, PD pathology displays the clear tropism of neurodegeneration in dopaminergic neurons, such as the SN neurons.
As a factor promoting this αSyn pathology, posttranslational modifications (PTMs) of αSyn have been implicated, such as phosphorylation, ubiquitination, SUMOylation, or truncation, which alter characteristics of αSyn and affect the conformation of aggregates [18][19][20][21] . The most studied PTM is phosphorylation at Ser129 (pS129) that is found on more than 90% of the αSyn in Lewy bodies from PD patients and considered an important marker for synucleinopathy 18,22 . Although the specific function of pS129 remains unclear, it seems to promote formation of αSyn filaments as well as oligomers 23,24 . However, since the multiple PTMs are mediated by the ubiquitous enzymes localized throughout the entire brain, they do not completely explain the tropism of PD pathology.
The susceptible target in PD is a catecholamine-containing neuron in the SN, Locus coeruleus or sympathetic ganglia, where tyrosine hydroxylase (TH) is specifically expressed. TH is a rate-limiting enzyme of catecholamine biosynthesis and hydroxylates the side chain of tyrosine to form dihydroxyphenylalanine (DOPA) 25 . The functional interaction between αSyn and TH has been reported, in which αSyn regulates the dopamine biosynthesis by reducing TH phosphorylation and its enzymatic activity [26][27][28] . However, the relationship of TH activity to αSyn aggregation kinetics or αSyn pathology in PD is still unexplored. The overlap of PD pathology with TH distribution led us to consider the possibility of TH functioning as an αSyn PTM enzyme. In this study, we show that TH hydroxylates αSyn at Tyr136 in vitro and in vivo, which facilitates oligomer formation and induces neurotoxicity, suggesting that TH contributes to PD pathogenesis.

Identification of TH-mediated αSyn hydroxylation
To examine whether TH catalyzes the hydroxylation of tyrosine residues in αSyn, we first performed an in vitro reaction assay ( Supplementary Fig. 1a) using recombinant TH (enzymatic activity: 624 units/mg) 29 with wild-type αSyn or two mutant forms of familial PD, E46K and A53T, characterized by promoting fibril formation 30,31 . After the reaction, αSyn was purified by reverse-phase high-performance liquid chromatography (RP-HPLC). The separation profiles of THtreated αSyn differed from those of untreated αSyn (control αSyn) in the appearance of an additional peak ( Supplementary Fig. 1b). We collected αSyn fractions, including the additional peak, and digested them to make small fragments for mass spectrometry (MS). Tryptic digestion provided two tyrosine-containing fragments, namely, residues 35-43 (Tyr39) and 103-140 (Tyr125, 133, and 136) of αSyn (Fig. 1a), which were separated by RP-HPLC. The resulting separation profiles displayed two corresponding peaks in control αSyn, while TH-treated αSyn introduced several new peaks ( Supplementary Fig. 2a). The larger fragment, residues 103-140, was subsequently digested by Asp-N, which provided three tyrosine-containing fragments: residues 119-125 (Tyr125), 126-134 (Tyr133), and 135-140 (Tyr136) (Fig. 1a). These fragments were also separated by RP-HPLC ( Supplementary Fig. 2b). THtreated αSyn also displayed different separation profiles accompanied by additional new peaks, implying the occurrence of some modification by TH treatment (Supplementary Fig. 2b).
We  a Digestion process of αSyn by trypsin (upper) and Asp-N (lower). Trypsin digestion yielded two tyrosine (red)-containing fragments (blue), whose sequences were boxed. A fragment of residues 103-140 of αSyn (103-140 aa) was further digested by Asp-N, yielding five fragments, as shown by the boxed sequences. Here, Asp-N atypically cleaves the N-terminus of Glu126 but fails at N-terminus of Asp121. b MALDI-TOF mass spectrum of the fragment corresponding to 135-140 aa. The peak at m/z 723.19 corresponds to 135-140 aa (control, left). After TH treatment (right), an additional peak at m/z 739.18 showed an increment of 16 mass units compared to that of the 135-140 aa fragment. The peak at m/z 745.2 corresponds to sodium adduct form of the 135-140 aa. c MALDI-TOF MS/MS spectrum of the MS peak at m/z 723. 2 (upper) or m/z 739.2 (lower) in the TH-treated sample derived from (b). Fragmentation sites are indicated in the boxed area. The peptide sequences of fragmentated ions are shown above each peak, identifying the site of dopanization as Tyr136.
Figs. 2c and 4a, b). Amino acid sequencing by MALDI-TOF MS/MS analysis at m/z 739.2 showed that Tyr136 gained 16 mass units, indicating a conversion of Tyr136 to DOPA (Y136DOPA) (Fig. 1c and Supplementary Figs. 2c and 4c, d). Concurrently, we confirmed that there was no oxidation at the C-terminal methionine Met127, which was susceptible to reactive oxygen species (ROS) produced through an oxidative reaction by TH ( Supplementary Fig. 3c). These data indicate that TH catalyzes the dopanization of the intraprotein tyrosine residue Tyr136 in αSyn.

Hydroxylation of overexpressed αSyn by endogenous TH in vivo
To obtain insights into the function of dopanized αSyn, we generated a mouse monoclonal antibody against Y136DOPA (Y136DOPAmab; Supplementary Fig. 5a) and confirmed its specificity ( Fig. 2a and Supplementary Fig. 5b). Using this antibody, we first checked the modification rates at which TH converted approximately 40% of αSyn to Y136DOPA in the in vitro reaction ( Supplementary Fig. 5c, d). To explore the dopanization of αSyn in cultured cells, we introduced mCherry (mCh)-αSyn using lentivirus-mediated gene transfer into primary sympathetic neurons isolated from the superior cervical ganglion (SCG) of mice in which endogenous TH was highly expressed. Western blot (WB) analysis using the Y136DOPAmab indicated a weak signal at Day 4 after viral infection, which was strengthened at Day 8 ( Fig. 2b). Similar results were obtained for TH-positive neuronal PC12 cells overexpressing mCh-αSyn ( Fig. 2c and Supplementary Fig. 6a). Immunofluorescence also indicated the Y136DOPA modification of exogenous mCh-αSyn in PC12 cells ( Fig. 2d and Supplementary  Fig. 6b). Furthermore, we examined the dopanization of mCh-αSyn that was overexpressed in mouse SN neurons by stereotaxic injection of adeno-associated virus (AAV; Supplementary Fig. 6c). Y136DOPA was also detected at 5 weeks after injection by WB as well as immunohistochemistry, which labeled both the cell bodies of SN neurons and their nerve terminals in the striatum (Fig. 2e, f and Supplementary  Fig. 6d-f). To examine whether this in vivo dopanization was catalyzed by endogenous TH, PC12 cells expressing mCh-αSyn were incubated with a TH inhibitor, α-methyl-p-tyrosine (AMPT), and analyzed by WB. After 9 hours (h) of treatment, the Y136DOPA signal was markedly decreased compared to that of inhibitor-untreated cells ( Fig. 2g and Supplementary Fig. 6g), indicating that TH hydroxylation activity is required for Y136DOPA modification in vivo. In addition, we also investigated the Ser129 phosphorylation (pS129) of αSyn, a major PTM associated with αSyn pathology. Immunocytochemistry showed that the pS129 modification of mCh-αSyn overlapped with the Y136DOPA signals in PC12 cells ( Supplementary Fig. 6h), suggesting that dopanization is involved in the onset of αSyn pathology. Collectively, overexpressed αSyn was posttranslationally dopanized at Tyr136 by endogenous TH in vivo.

Dopanization of αSyn in human synucleinopathy
Next, we examined the Y136DOPA modification in a mouse model of PD. We used heterozygous A53T BAC transgenic (Tg) mice that express human A53T similarly to the endogenous αSyn expression pattern and display the degeneration of SN neurons in an age-dependent manner 32 . Immunohistochemistry staining using Y136DOPAmab did not indicate any signals in the cell bodies at SN pars compacta (SNc) from either 18month-old wild-type or Tg mice (Fig. 3a). In contrast, the striatum to which SN neurons were projecting showed punctate signals in Tg mice but not in wild-type mice (Fig. 3a). Furthermore, a similar analysis was performed on brain sections from patients with α-synucleinopathies, PD and multiple system atrophy (MSA), revealing a significant increase neurons. mCh-αSyn was strongly overexpressed in the SNc by the stereotaxic injection of AAV. WB using the SN tissue detected Y136DOPA at 5 weeks after injection. f Immunohistochemistry staining of AAV-injected brain sections. Y136DOPA modification (green) of mCh-αSyn (red) was detected in the cell bodies of TH-positive dopaminergic neurons (blue) in the SNc (upper, white arrowheads), as well as their nerve terminals (white) in the striatum (lower, blue arrows). Scale bars: 20 µm (upper) and 10 µm (lower). g Y136DOPA signals decreased after treatment with the TH inhibitor. PC12 cells expressing mCh-αSyn at d10 after lentiviral infection were incubated with AMPT for 9 h and harvested. WB showed that the Y136DOPA signals were reduced after inhibiting TH enzyme activity. Data are representative of two independent experiments with similar results (a-g).
in the Y136DOPA-positive area in the posterior putamen of patients compared with that of the control sections (Fig. 3b, c). These signals were colocalized with the TH-positive axon terminals of the SN neurons in the putamen (Fig. 3d), indicating that dopanized αSyn was distributed in the presynapses of dopaminergic neurons in PD and MSA cases. These results suggest that TH-mediated Y136DOPA modification is involved in the pathogenesis of human αsynucleinopathies.
Oligomer assembly of αSyn triggered by Y136DOPA modification An increasing amount of evidence suggests that the oligomeric and fibrillar aggregation of αSyn plays a central role in the pathogenesis of PD and other synucleinopathies 33 . In particular, the oligomeric form of αSyn has a higher cytotoxicity, leading to the disruption of biological membrane integrity 15 . To examine the effects of Y136DOPA modification on fibrillization, recombinant FLAG-tagged αSyn (f-αSyn, f-E46K, or f-A53T) with or without prior TH-mediated dopanization was incubated with continuous agitation for 18 h. After incubation, we confirmed that both control and dopanized f-αSyn were detected by an antibody, Syn-O2, that recognized both oligomeric and fibrillar conformations of αSyn 34 in a dot blot assay ( Supplementary Fig. 7a). A solubility assay using these oligomeric/fibrillar f-αSyn showed that sarkosyl-insoluble f-αSyn aggregates were reduced by TH treatment ( Supplementary Fig. 7b). We then examined the structures of f-αSyn by transmission electron microscopy (TEM) after 18 h of incubation and found that control f-αSyn formed large fibril aggregates. On the other hand, to our surprise, dopanized f-αSyn formed short, dispersed oligomer-like structures (Fig. 4a). Similarly, dopanized f-E46K and f-A53T also formed separated short oligomer-like assemblies (Supplementary Fig. 7c). These data suggest that the dopanization of Tyr136 prevents the assembly of large aggregates and stabilizes small oligomer-like conformations. We further used sucrose gradient centrifugation to estimate the molecular weight of the f-αSyn assemblies. Before 18 h of incubation, both control and dopanized f-αSyn displayed a small molecular mass regardless of TH treatment ( Fig. 4b and Supplementary Fig. 7d). After 18 h of incubation, control f-αSyn formed aggregates with higher molecular masses, while dopanized f-αSyn still retained smaller masses, similar to the TEM images ( Fig. 4b and Supplementary Fig. 7d). To confirm the oligomer formation of small-mass dopanized f-αSyn, we performed size exclusion chromatography (SEC) and compared the results with those of monomeric recombinant αSyn. The resulting chromatograms showed that dopanized f-αSyn formed oligomers up to 15-mers after 18 h of incubation ( Fig. 4c and Supplementary Fig. 7e). These data support that Y136DOPA modification of αSyn promotes the maintenance of a small oligomeric state and prevents the formation of large-mass aggregation.

Cytotoxicity of dopanized αSyn
Cell-to-cell propagation of αSyn aggregates in a prion-like manner is thought to contribute to the progression and spreading of αSyn neuropathology in synucleinopathy 35 . We investigated the cellular uptake and toxicity of dopanized f-αSyn after 18 h of incubation using PC12 cells. At 24 h after the addition of f-αSyn to the culture medium, dopanized f-αSyn was internalized into cells as well as control f-αSyn ( Fig. 4d and Supplementary Fig. 8a). Then, we examined the cytotoxicity of f-αSyn by the real-time quantification of cell death using the IncuCyte Live-Cell Analysis System. PC12 cells treated with dopanized f-αSyn exhibited a significant increase in cell death in a timedependent manner compared to that of cells treated with control f-αSyn ( Fig. 4e and Supplementary Fig. 8b). Collectively, these results demonstrate that dopanized αSyn has a higher neurotoxicity than control αSyn, suggesting that dopanized αSyn and its oligomer species may contribute to the selective loss of dopaminergic neurons in the PD brain.

Discussion
In this study, we discovered a PTM of αSyn in which TH converts Tyr136 to DOPA in SN neurons. Although TH has been broadly recognized as an enzyme for the hydroxylation of free tyrosine molecules 36 , our results indicate the possibility that the intraprotein tyrosine residue is also an enzymatic substrate for TH. Catalytic domains of enzymes are mainly buried inside the protein tertiary structure to ensure their substrate specificities 37 . However, the catalytic iron atom of TH is located near the surface, 10 Å below the enzyme surface within a 17 Å-deep active-site cleft 38 ( Supplementary  Fig. 9a). Grid-based HECOMi finder (GHECOM) calculations also show the presence of a large groove containing the catalytic site on the TH surface ( Supplementary Fig. 9a). On the other hand, the crystal structure of αSyn shows that Tyr136 is located in its flexible C-terminal tail ( Supplementary Fig. 9b). This intrinsically disordered (ID) C-terminal region does not form a three-dimensional structure under physiological conditions and even in fibril constructs, likely allowing Tyr136 to fit into the TH catalytic site 39 . In contrast, Tyr39 located between the two α-helices probably does not become a substrate for TH due to the structural restriction. ID domains in proteins are generally susceptible to PTM 40,41 , which changes their physicochemical properties and induces the alteration of their conformation. Similarly, Y136DOPA modification caused significant changes in αSyn aggregation kinetics. Based on previous works and our findings, we speculate that other proteins with flexible ID domains may be also subjected to TH-mediated hydroxylation for modifying their biochemical properties. Further study is needed to confirm the generality of dopanization as a PTM.
In the in vitro reaction assay and MS analysis, we were unable to detect dopanization at two tyrosine residues in the C-terminal region, Tyr125 and Tyr133, despite their proximity to Tyr136. This result may imply the presence of a preferential sequence for dopanization. Another possibility is that the lower stability of DOPA-containing fragments under the experimental conditions might make MS detection difficult. Catechol compounds are prone to releasing ROS in the presence of transition metals and oxygen, which induces nonenzymatic peptide degradation 42,43 . Indeed, the RP-HPLC peak corresponding to C-terminal region of αSyn showed a remarkable reduction in height after TH treatment (Supplementary Fig. 2a). Considering the influence of ROS from the "DOPA residue" on peptide stabilities, the possibility of dopanization at Tyr125 and Tyr133 is not completely precluded.
Amyloid aggregation of αSyn in the SN is closely associated with the selective loss of dopaminergic neurons, resulting in defective physical movements 44,45 . However, recent findings have demonstrated that oligomeric αSyn species exacerbate αSyn-mediated toxicity 46,47 . Moreover, the injection of sonicated short αSyn fibrils into mouse striatum propagates Lewy pathology and results in the progressive loss of dopaminergic neurons 35 . The Y136DOPA modification of αSyn induced the formation of an oligomeric structure and displayed a higher cytotoxicity to PC12 cells than the control. Some PTMs, such as C-terminal truncation and phosphorylation, have been implicated in αSyn oligomer formation and neurotoxicity 18,21 . The negatively charged C-terminus and phosphorylation at Ser129 may act to retard fibril formation with a β-sheet amyloid structure 48,49 . Presumably, the dopanization of αSyn at Tyr136 augments its hydrophilicity and thereby triggers the formation of oligomers, which may play a pathogenic role in dopaminergic neurodegeneration. The Y136DOPA signal was also detected at the striatonigral axon terminals of aged A53T Tg mice and patients with PD and MSA. These results strongly suggest the involvement of αSyn dopanization in pathology in vivo. On the other hand, neither control mice nor control human brains showed any dopanized αSyn signal, which may suggest the possibility that the dopanized αSyn is physiologically undetectable and accumulates depending on pathological progression. Altogether, our findings implicate that TH participates in αSyn pathology, providing a potential explanation for the selective loss of dopaminergic neurons in synucleinopathies.
The familial PD-linked pathogenic mutations act to accelerate disease onset and progression 4,5,50 . In the fibrillization assay, we found no obvious differences in the effect of dopanization among wild-type and mutated αSyn in this study. Previous works have revealed that E46K and A53T accelerate the formation of large fibrillar aggregates 30,31 . Consistently, control E46K and A53T without TH treatment formed aggregates with larger masses compared to that of the wild-type, whereas the dopanization made wild-type αSyn and two mutants assemble into the short oligomeric forms ( Fig. 4b and Supplementary Fig. 7d). Presumably, the increased hydrophilicity caused by dopanization may contribute to abolishing this aggregation preference in mutated αSyn, suggesting that dopanization is a critical factor in promoting oligomer formation. In addition to point mutations, the increases in αSyn protein levels and aging has also been considered a major factor in PD, which promotes the abnormal accumulation of misfolded and stabilized αSyn [51][52][53] . Altogether, the aberrant metabolism of αSyn forms the foundation of PD, and the dopanization of αSyn may trigger the preferential degeneration of dopaminergic neurons. Further analysis may help us to precisely explain how this new PTM contributes to PD pathogenesis. Chemical compounds that protect the dopanization of αSyn but do not suppress free DOPA synthesis by TH may be applicable in preventing the progression of PD. Our work will open the way to drug discovery for the therapeutic intervention of PD.

Methods
All methods were performed in accordance with the approved guidelines. All animal studies were approved by the Institutional Animal Care and Use Committee, Osaka Metropolitan University (#21094), and Kyoto University (#Med Kyo 21001). Recombinant DNA experiments were approved by the Safety Committee for Experiments using Living Modified Organism, Osaka Metropolitan University (#708). Experiments with human samples were approved by the Ethics Committee of Kyoto University (#R1038).

In vitro reaction of αSyn and TH
The in vitro reaction of TH (10 µM) and αSyn or two mutants (5 µM each) was conducted in 20 mM MES, pH 6.2, 100 µM BH 4 , and 25 µM FeSO 4 in a 50 µL reaction volume. Incubation was performed for 10 min at 37°C. The negative control described as 'Control' in this study contained neither TH nor BH 4 . For RP-HPLC and fibrillization, GST-αSyn and GST-f-αSyn were used, respectively, and separated from the reaction mixture using GST-Sepharose. Binding to beads in 150 mM KCl and cleavage by PreScission protease were performed as described above.

RP-HPLC and MS analysis
The above αSyn and its mutants were purified by using RP-HPLC (L-7100, Hitachi LaChrom HPLC System). The proteins were loaded on a column (Develosil C-4 HG column, Nomura Chemical) that was equilibrated with 95% solvent A (0.1% trifluoroacetic acid (TFA)-H 2 O)/ 5% solvent B (0.1% TFA-acetonitrile (ACN)) and eluted using a linear gradient of solvent B (5-80%) in 40 min at a flow rate of 1 mL/min. Detection was performed by UV absorption at 220 nm. The eluted αSyn fraction was dried by a vacuum centrifugal concentrator, dissolved in 100 mM ammonium carbonate and 10% ACN, and digested with 1 pmol/µL trypsin (Promega). The digested products were subjected to a second RP-HPLC (HP1100 HPLC system, Agilent Technologies) equipped with a ZORBAX 300SB-C8 (Agilent Technologies), and eluted using the same elution gradient as the first RP-HPLC in 20 min at a flow rate of 0.32 mL/min. Detection was performed by UV absorption at 280 nm. The fraction containing the C-terminal fragment (residues 103-140) of αSyn was dried and digested by 4 ng/µL Asp-N (Roche), followed by a third RP-HPLC separation using the same chromatographic conditions and column as the second RP-HPLC separation. Eluted 0.5-min fractions were dried and dissolved in 10% ACN for MS analysis.
Each fraction was spotted onto a stainless steel plate, dried, and mixed with a matrix solution (0.5 µL per spot; 7 mg/mL of α-cyano-4hydroxycinnamic acid (Sigma) in 50% ACN, 0.1% TFA). MALDI-TOFMS and MS/MS measurements were carried out with a 4700 MALDI-TOF/ TOF mass spectrometer (Applied Biosystems) as previously described 57 . All mass spectra were obtained by averaging 2500 laser shots from each sample well in positive-ion mode. The instrument was calibrated with a mixture of peptides, angiotensin I (m/z 1296.6), dynorphin (m/z 1604.0), ACTH (1-24) (m/z 2932.6), and β-endorphin (m/z 3463.8). For the MS/MS measurements, the metastable suppressor and CID gas were both set to "ON". The entire process was controlled using 4000 series Explorer software (version 3.6; Applied Biosystems). Spectra were processed and analyzed using Data Explorer software (version 4.8; Applied Biosystems).

Generation of mouse monoclonal antibody
Peptide corresponding to the C-terminal residues 131-140 of human αSyn with additional cysteine at its N-terminus (Cys-EGYQDYEPEA, 136Y peptide), antigen peptide with DOPA instead of Tyr136 (Cys-EGYQD-Dopa-EPEA, 136DOPA peptide) and keyhole limpet hemocyanin (KLH)-conjugated 136DOPA peptide (KLH-136DOPA) were synthesized by Peptide Institute, Inc. Immunogen was prepared by mixing 2 mg/mL KLH-136DOPA with an equal volume of TiterMax Gold (TiterMax) to form a stable emulsion. We immunized twenty 7-weekold female BALB/c mice with the injection of immunogen emulsion subcutaneously into both hind footpads and back skin (total 100 µg/ mouse). Mice were maintained at 25°C with 55% humidity on a 12-h light-dark cycle, given free access to food and drinking water, and handled according to the institutional guideline for animal experiments. After 2 weeks, mice were subjected to the second immunization with an emulsion of KLH-136DOPA (50 µg/mouse) with Freund's incomplete adjuvant (Wako). After 1 week, a small amount of serum was taken from each immunized mouse to test the immune response against the 136DOPA peptide by ELISA. At 3 weeks after the second immunization, animals were administered 1 mg/mL KLH-136DOPA in PBS, pH 7.4, intraperitoneally as the final boost (150 µg/ mouse), followed by hybridoma generation at 4 days after booster injection.
Prior to hybridoma generation, mouse myeloma cells (Sp2/O-Ag14, JCRB9084, JCRB Cell Bank) were maintained in a 75 cm 2 flask with GIT medium, 10% FBS, and P/S. The spleen of immunized mice was dissected and dissociated from the capsule. Splenocytes and mouse myeloma cells were fused at a ratio of 5:1 by the addition of 1 mL of PEG 1500 (Roche) gently over 1 min with agitation. After incubation for 1 min, 15 mL of DMEM was slowly added over 5 min. Then, cells were collected and resuspended in HAT conditioned medium (GIT, 100 µM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine, 5% BM Condimed H1 (Roche) and P/S), spread in 96-well plates (2 × 10 5 splenocytes/well) and cultured for 9 days in a CO 2 incubator at 37°C before ELISA.

Enzyme-linked immunosorbent assay (ELISA)
For hybridoma screening, we diluted 136Y and 136DOPA peptide to 3 µg/mL with PBS and added 50 µL/well of this solution to 96-well immunoplates (Thermo Scientific), followed by the incubation for 2 h at 37°C. After washing with PBS, blocking buffer (0.5% BSA in PBS) was added to each well and incubated for 1 h at 37°C. After washing plates with PBS, hybridoma culture supernatant was applied onto each plate precoated by 136Y or 136DOPA peptides and incubated overnight at For quantification of dopanized αSyn in the in vitro reaction, a serial dilution of 136DOPA peptide (4,8,16, or 32 nM in PBS) was subjected to ELISA using Y136DOPAmab. After stopping the reaction, the OD at 490 nm was measured, and a standard curve was established.
Control and TH-treated αSyn after the in vitro reaction were purified by RP-HPLC, diluted at concentrations of 24 or 48 nM, and tested by ELISA using Y136DOPAmab. The amount of dopanized αSyn at Tyr136 by TH was calculated based on the standard curve.

Lentiviral packaging
Lentiviral vectors were produced as described previously 11 . Briefly, Lenti-X-293T cells (632180, Takara) were prepared on collagen-coated dishes with DMEM, 10% FBS, and P/S. The following day, cells were cotransfected with the three plasmids described above using polyethyleneimine (Sigma). After 48 h, culture medium containing the virus particles was harvested, centrifuged at 2000 × g for 10 min, filtered through 0.45-μm filters (Millipore) to remove cell debris, and concentrated by ultracentrifugation (Himac, Hitachi) using a swing rotor at 52,000 × g for 2 h. The virus particles were resuspended in sterile PBS and stored at −80°C until use.

Cell culture
For sympathetic neuron primary culture, superior cervical ganglions (SCGs) were dissected from twenty postnatal day 0-2 male or female C57BL/6 mice (ten mice/experiment) and transferred to L15 medium. After centrifugation, SCGs were incubated with 1 mg/mL collagenase at 37°C for 1 h, followed by treatment with 0.25% trypsin and 0.1% DNase at 37°C for 20 min. After trituration, cells were spread on collagen-coated 12-well plate (cells from 5 ganglions/well) with DMEM/Ham's F12 medium supplemented with N-2 supplement (Invitrogen), 100 ng/mL murine NGF (Promega), GlutaMAX (Invitrogen) and P/S. Half the volume of culture medium was exchanged with fresh medium every 2 days. At 2 days in vitro (DIV), the lentiviruses carrying mCh-αSyn were added to the SCG culture for overexpression. To prevent glial proliferation, cytosine-1-β-D-arabinofuranoside (AraC, Sigma) was added at a 0.25 µM concentration to cultures at DIV 4. SCGs were harvested sequentially at DIV 6 or 10 (Days 4 or 8 after viral infection, respectively) and subjected to WB analysis.

AAV-stereotaxic injection and immunohistochemistry
AAV serotype 9 vectors for the expression of GFP, mCh-αSyn, or its mutants were produced as described previously 58 . Anesthetized sixteen 9-week-old male or female C57BL/6 mice (four mice/AAV) were placed in a stereotaxic frame. AAV solutions at a volume of 0.7 µL were injected through a glass capillary, unilaterally targeting the left SN at the following coordinates: 1.3 mm rostral relative to lambda, 1.5 mm lateral, and 4.3 mm ventral. The glass capillary was left in place for 5 min before it was slowly retracted.
Three weeks after surgery, mice were transcardially perfused with Zamboni solution (2% PFA, 0.2% picric acid in 0.1 M PB, pH7.4). Brains were removed, postfixed in Zamboni solution overnight, and placed in 30% (w/v) sucrose in 0.1 M PB for 2 days, followed by freezing in powdered dry ice. Coronal sections of the SN and striatum were cut using a cryostat (CM1950, Leica Microsystems) at 16-µm thickness. For immunohistochemistry, sections were dried and washed with PBS. Antigen retrieval was carried out in 10 mM citric buffer, pH 6.0, at 80°C for 20 min. After blocking with 1% BSA and 0.3% Triton X-100 in PBS for 1 h, sections were incubated with mouse Y136DOPAmab and rabbit anti-TH antibody (1:1000, Millipore, AB152) overnight at 4°C. After washing with PBS, sections were incubated with Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor 488 (1:1000), Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488 (1:1000, Invitrogen, A11008), Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor 647 (1:1000) and DAPI (1:3000) in blocking buffer at RT for 2 h. After rinsing with PBS, sections were mounted. Microscope images were acquired using LSM700 confocal microscopy and ZEISS ZEN software.

Immunohistochemistry of transgenic (Tg) mice
A53T BAC Tg mice were generated previously 32 . Three male or female C57BL/6 and Tg mice (18 months old) were anesthetized with sevoflurane and intracardially perfused with cooled PBS followed by 4% PFA. The brain samples were postfixed with 4% PFA and embedded in paraffin. Coronal sections of the SN and striatum were cut on a microtome (EG1150, Leica Microsystems) at 8-µm thickness. The sections were pretreated in 100% formic acid at RT for 15 min and then in 0.1 M PBS at 120°C for 10 min using an autoclave. For quenching endogenous peroxidase, sections were treated with 3% (v/v) H 2 O 2 in PBS for 30 min at RT. After blocking using mouse blocking reagent (414322, Histofine Mouse stain kit, Nichirei Biosciences) for 1 h at RT, sections were then incubated with Y136DOPAmab overnight at 4°C, followed by appropriate polymer secondary antibody (414322, Histofine mouse stain kit, Nichirei Biosciences). Between steps, the sections were washed three times with 0.1 M PBS for 5 min each and visualized using a peroxidase stain DAB kit (25985-50, Nakarai Tesque). The images of interest were captured with a microscope (BX43, Olympus).

Immunohistochemistry of human brain
Formalin-fixed human brain tissue samples from 3 patients with MSA (71-78 years old), 4 patients with PD (67-78 years old), and 7 agematched controls (62-86 years old) without significant αSyn disease and clinical history of parkinsonism were used in this study, as shown in Supplementary Table 1. For the study with autopsied materials, all the bereaved family signed the written informed consent, which includes that the donated human autopsied tissues and clinical information will be used for academic conferences and scientific paper presentations. To obtain the corresponding portion from each case, the posterior putamen was obtained from coronal sections containing the globus pallidus. Patient diagnosis was determined by clinical information and pathological examination.
For DAB staining, 6-µm paraffin sections were pretreated at 120°C for 20 min in Histofine deparaffinizing antigen retrieval buffer, pH 6 (415281, Nichirei Biosciences), using an autoclave, followed by overnight incubation with Y136DOPAmab in PBS containing 3% BSA at 4°C. The sections were incubated with peroxidase polymer secondary antibody (424154, Histofine Simple Stain MAX-PO MULTI, Nichirei Biosciences) and developed with a DAB Substrate Kit (SK 4100, Vector Laboratories). After immunostaining, sections were counterstained with hematoxylin and cover slipped. For quantification of Y136DOPAmab-positive area densities, pictures under 200× magnification for each slide to encompass the lateral part of the putamen from each case were taken. The positive area percentage was calculated for each picture automatically using Fiji software (https://fiji.sc/).
For the antibody absorption assay, Y136DOPAmab was preincubated with 1 µg/mL 136DOPA or 136Y peptides overnight at 4°C before use.
For the f-αSyn solubility assay, f-αSyn and its mutants after fibrillization were centrifuged at 627,000 × g (120,000 rpm, Beckman Optima MAX-TL ultracentrifuge with TLA 120.2 rotor) for 1 h. The pellet was dissolved in 100 µL of 2% sarkosyl, 10 mM MES, pH 6.2, and 150 mM KCl and centrifuged at 627,000 × g for 20 min. The pellet was dissolved in 50 µL of 10 mM MES, pH 6.2, and 150 mM KCl and spotted on nitrocellulose membranes (5 µL each) for the dot blot assay.
For the cytotoxicity assay, an IncuCyte live-cell analysis system (Sartorius) was applied to monitor cell death. PC12 cells were cultured in 96-well plate at 6000 cells/well. At DIV 4-5, cells were treated with 5 µL of above f-αSyn solution (final 0.7 µM) or PBS and IncuCyte Cytotox Green Dye at a concentration of 200 nM. Then, the plate was placed in an IncuCyte incubator at 37°C. Dead cells with green fluorescence were monitored and analyzed with IncuCyte ZOOM software according to the manufacturer's instructions. The data were obtained from distinct 3 wells per each sample at the same time.

Sucrose density gradient centrifugation
The f-αSyn after fibrillization prepared above (7 μM, 400 μL each) were layered on a 12 mL 5-30% (w/v) linear sucrose gradient in 20 mM HEPES, pH 7.4, 150 mM KCl, 0.1% NP-40 and 0.5 mM DTT and centrifuged at 290,000 × g for 24 h. The tube was then punctured at the bottom, and 12 of 1-mL fractions were collected. The fractions were examined by WB using a mouse anti-αSyn antibody.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
The data supporting the findings of this study are available in the manuscript and its supplementary information file. The TH and αSyn structure data used in this study are available in the Protein Data Bank under accession code PDB-1TOH and PDB-1XQ8, respectively. The statistics data generated in this study are provided in the Source data file. Source data are provided with this paper.